Project Summary: Mild traumatic brain injury (TBI), or concussion, is a growing public health concern, as large populations of athletes, soldiers, and other young people are exposed to high risks of repetitive head injuries and associated long-term risks of neurodegeneration. Early, accurate diagnosis of mild TBI is critical for effective intervention and proper recovery, but remains an unmet clinical need. Current diagnoses are time-consuming and rely on subjective clinical assessments and neuroimaging, which usually cannot show clear structural brain damage in mild TBI. This work proposes to engineer an objective, ultrasensitive point-of-care diagnostic tool that can rapidly determine the presence and severity of mild TBI at the point of injury, to enable early treatment and sufficient recovery times. The proposed platform will utilize single molecule detection to measure low abundance protein biomarkers for mild TBI in saliva, as a noninvasive test that can easily be deployed in schools, military fields, and emergency rooms. Single Molecule Array (Simoa) technology, developed by the Walt lab, is the current state of the art for ultrasensitive protein detection, using digital enzyme-linked immunosorbent assay (ELISA) to isolate and count single molecules captured on beads in femtoliter-sized microwells. Despite achieving 1000-fold higher sensitivity than conventional ELISA, the current Simoa technology cannot be used in point-of-care applications due to its significant cost, size, and need for specially trained personnel. The sensitivity of Simoa also remains insufficient for detecting brain-derived and other potential protein biomarkers that exist at levels below its detection limit (~10-15 M), particularly in minimally invasive biofluids such as plasma and saliva. To address these challenges, this work will design a simple, cost- effective, and more sensitive Simoa platform, by dropcasting beads in a monolayer film for single molecule counting, eliminating the need for costly microwells and bulky fluidics. Furthermore, this approach can greatly improve sampling of rare molecules over the current Simoa technology, which will significantly enhance sensitivity. This system will be integrated with a handheld reader as a point-of-care diagnostic tool and applied for multiplexed detection of a salivary protein biomarker panel for mild TBI. The point-of-care platform will be benchmarked against current Simoa assays for the biomarker panel and validated as a sensitive, specific diagnostic tool for mild TBI, using saliva samples from concussed and healthy athletes. This project will also provide rigorous scientific training for Dr. Connie Wu that will prepare her for a fully independent academic research career. She will work closely with Dr. David Walt in a highly interdisciplinary, collaborative environment at Brigham and Women?s Hospital and the Wyss Institute at Harvard University, and develop an extensive skillset in analytical chemistry, diagnostics, and technology development and translation to complement her previous training. This training plan also provides opportunities for Connie to expand her scientific expertise and professional skills through coursework, seminars, presentations at conferences, and grant writing workshops.